Nonmuscle myosin heavy chain (NMHC) II is encoded by three different genes in mammals (Myh9, Myh10 and Myh14). The nonmuscle myosin (NM) II proteins, which include dimers of one isoform of these heavy chains, along with two pairs of light chains, are named NM II-A, NM II-B, and NM II-C. These myosin isoforms are widely distributed in mammalian tissue and in all cell lines. The relative amounts of the three myosins are important in order to interpret the phenotypes in null mice and the effects of siRNA mediated decreases of specific isoforms. There is no direct way of easily determining these values currently available. Antibody staining of specific isoforms is useful, but not quantitative due to the disparate nature of antigen recognition. The use of mass spectroscopy (MS) provides a direct method to estimate the amounts of the different myosin heavy chain isoforms from extracts of various tissues and cells, which have been separated by SDS-polyacrylamide gel electrophoresis. NMHC II bands (230kDa) are excised from Coomassie blue stained gels, destained, reduced and alkylated, subjected to tryptic digestion and analyzed by liquid chromatography tandem MS. To test the method, we used RBL cells which lack NM II-B and Cos-7 cells, which do not contain NM II-A. The MS results indicate that NMHC IIs from RBL cells are 98% II-A, <1% II-B and II-C; NMHC IIs from Cos-7 cells are 86% II-B, 14% II-C, and II-A is undetectable. The same method was applied to tissues, which are a mixture of different cell types such as spleen where the absence of NM II-B and II-C by immunoblot analysis was confirmed by MS. Our results show that MS can be a useful tool for estimating content of NM II isoforms in tissues and cells and for interpreting experimental interventions such as siRNA.